HighALPS: ultra-high-throughput marker-gene amplicon library preparation and sequencing on the Illumina NextSeq and NovaSeq Platforms

Abstract

Marker gene amplicon sequencing on Illumina devices remains the most commonly used technology to profile microbial communities. Yet, most library preparation protocols are not adapted to harness the capabilities and deal with the caveats of the latest Illumina sequencing platforms, which highly outperform older platforms in terms of speed, quality, and output. Here, we present an ultra-high-throughput, cost-effective, and robust library preparation protocol (HighALPS) optimized to fully leverage the capabilities of the latest Illumina sequencing platforms. The combinatorial unique dual index strategy effectively combats miss-assignment of reads due to index-hopping, which is more prevalent in newer platforms. The HighALPS protocol incorporates technological (e.g., novel sequencing chemistry and lab automation platforms) as well as bioinformatics advances (e.g., denoising algorithms which make triplicate amplifications unnecessary) of the last few years to optimize and streamline library preparation for bacterial and fungal communities.